Papanicolaou test for ovarian and endometrial cancers

ABSTRACT

The recently developed liquid-based Papanicolaou (Pap) smear allows not only cytologic evaluation but also collection of DNA for detection of HPV, the causative agent of cervical cancer. We tested these samples to detect somatic mutations present in rare tumor cells that might accumulate in the cervix once shed from endometrial and ovarian cancers. A panel of commonly mutated genes in endometrial and ovarian cancers was assembled and used to identify mutations in all 46 endometrial or cervical cancer tissue samples. We were able also able to identify the same mutations in the DNA from liquid Pap smears in 100% of endometrial cancers (24 of 24) and in 41% of ovarian cancers (9 of 22). We developed a sequence-based method to query mutations in 12 genes in a single liquid Pap smear without prior knowledge of the tumor&#39;s genotype.

This invention was made with government support under grant no.CA043460, CA062924, CN043309, and CA129825 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

TECHNICAL FIELD OF THE INVENTION

This invention is related to the area of cancer screening. Inparticular, it relates to ovarian and endometrial cancers.

BACKGROUND OF THE INVENTION

Since the introduction of the Papanicolaou test, the incidence andmortality of cervical cancer in screened populations has been reduced bymore than 75% (1, 2). In contrast, deaths from ovarian and endometrialcancers have not substantially decreased during that same time period.As a result, more than 69,000 women in the U.S. will be diagnosed withovarian and endometrial cancer in 2012. Although endometrial cancer ismore common than ovarian cancer, the latter is more lethal. In the U.S.,approximately 15,000 and 8,000 women are expected to die each year fromovarian and endometrial cancers, respectively (Table 1). World-wide,over 200,000 deaths from these tumors are expected this year alone (3,4).

In an effort to replicate the success of cervical cancer screening,several approaches for the early detection of endometrial and ovariancancers have been devised. For endometrial cancers, efforts have focusedon cytology and transvaginal ultrasound (TVS). Cytology can indeedindicate a neoplasm within the uterus in some cases, albeit with lowspecificity (5). TVS is a noninvasive technique to measure the thicknessof the endometrium based on the fact that endometria harboring a cancerare thicker than normal endometria (6). As with cytology, screeningmeasurement of the endometrial thickness using TVS lacks sufficientspecificity because benign lesions, such as polyps, can also result in athickened endometrium. Accordingly, neither cytology nor TVS fulfillsthe requirements for a screening test (5, 7).

Even greater efforts have been made to develop a screening test forovarian cancer, using serum CA-125 levels and TVS. CA-125 is a highmolecular weight transmembrane glycoprotein expressed by coelomic- andMüllerian-derived epithelia that is elevated in a subset of ovariancancer patients with early stage disease (8). The specificity of CA-125is limited by the fact that it is also elevated in a variety of benignconditions, such as pelvic inflammatory disease, endometriosis andovarian cysts (9). TVS can visualize the ovary but can only detect largetumors and cannot definitively distinguish benign from malignant tumors.Several clinical screening trials using serum CA-125 and TVS have beenconducted but none has shown a survival benefit. In fact, some haveshown an increase in morbidity compared to controls because falsepositive tests elicit further evaluation by laparoscopy or exploratorylaparotomy (10-12).

Accordingly, the U.S. Preventative Services Task Force, the AmericanCancer Society, the American Congress of Obstetricians andGynecologists, as well as the National Comprehensive Cancer Network, donot recommend routine screening for endometrial or ovarian cancers inthe general population. In fact, these organizations warn that “thepotential harms outweigh the potential benefits” (13-16). An exceptionto this recommendation has been made for patients with a hereditarypredisposition to ovarian cancer, such as those with germline mutationsin a BRCA gene or those with Lynch syndrome. It is recommended that BRCAmutation carriers be screened every 6 months with TVS and serum CA-125,starting at a relatively early age. Screening guidelines for women withLynch syndrome include annual endometrial sampling and TVS beginningbetween age 30 and 35 (15, 17).

The mortality associated with undetected gynecologic malignancies hasmade the development of an effective screening tool a high priority. Animportant observation that inspired the current study is thatasymptomatic women occasionally present with abnormal glandular cells(AGCs) detected in a cytology specimen as part of their routine cervicalcancer screening procedure. Although AGCs are associated withpremalignant or malignant disease in some cases (18-22), it is oftendifficult to distinguish the AGCs arising from endocervical, endometrialor ovarian cancer from one another or from more benign conditions. Thereis a continuing need in the art to detect these cancers at an earlierstage than done currently.

SUMMARY OF THE INVENTION

According to one aspect of the invention a method is provided fordetecting or monitoring endometrial or ovarian cancer. A liquid Papsmear of a patient is tested for agenetic or epigenetic change in one ormore genes, mRNAs, or proteins mutated in endometrial or ovarian cancer.Detection of the change indicates the presence of such a cancer in thepatient.

According to another aspect of the invention a method is provided forscreening for endometrial and ovarian cancers. A liquid Pap smear istested for one or more mutations in a gene, mRNA, or protein selectedfrom the group consisting of CTNNB1, EGFR, PI3KCA, PTEN, TP53, BRAF,KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2, RFF43, andFGFR2. Detection of the mutation indicates the presence of such a cancerin the patient.

Another aspect of the invention is a kit for testing a panel of genes inPap smear samples for ovarian or endometrial cancers. The kit comprisesat least 10 probes or at least 10 primer pairs. Each probe or primercomprises at least 15 nt of complementary sequence to one of the panelof genes. At least 10 different genes are interrogated. The panel isselected from the group consisting of CTNNB1, EGFR, PI3KCA, PTEN, TP53,BRAF, KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2,RFF43, and FGFR2.

Still another aspect of the invention is a solid support comprising atleast 10 attached probes. Each probe comprises at least 15 nt ofcomplementary sequence to one of a panel of genes, wherein the panel isselected from the group consisting of CTNNB1, EGFR, PI3KCA, PTEN, TP53,BRAF, KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2,RFF43, and FGFR2.

Another aspect of the invention is a solid support comprising at least10 primers attached thereto. Each primer comprises at least 15 nt ofcomplementary sequence to one of a panel of genes. The panel is selectedfrom the group consisting of CTNNB1, EGFR, PI3KCA, PTEN, TP53, BRAF,KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2, RFF43, andFGFR2.

These and other embodiments which will be apparent to those of skill inthe art upon reading the specification provide the art with methods forassessing ovarian and endometrial cancers in a screening environmentusing samples that are already routinely collected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Schematic demonstrating the principle steps of the proceduredescribed in this study. Tumors cells shed from ovarian or endometrialcancers are carried into the endocervical canal. These cells can becaptured by the brush used for performing a routine Pap smear. The brushcontents are transferred into a liquid fixative, from which DNA isisolated. Using next-generation sequencing, this DNA is queried formutations that indicate the presence of a malignancy in the femalereproductive tract.

FIG. 2 . Diagram of the assay used to simultaneously detect mutations in12 different genes. A modification of the Safe-SeqS (Safe-SequencingSystem) protocol, for simultaneous interrogation of multiple mutationsin a single sample, is depicted. In the standard Safe-SeqS procedure,only one amplicon is assessed, while the new system is used to assessmultiple amplicons from multiple genes at once.

FIG. 3 . Mutant allele fractions in Pap smear fluids. The fraction ofmutant alleles from each of 46 pap smear fluids is depicted. The stageof each tumor is listed on the Y-axis. The X-axis demonstrates the %mutant allele fraction as determined by Safe-SeqS.

FIG. 4 . Heat map depicting the results of multiplex testing of 12 genesin Pap smear fluids. Each block on the y-axis represents a 30-bp blockof sequence from the indicated gene. The 28 samples assessed (14 fromwomen with cancer, 14 from normal women without cancer) are indicated onthe x-axis. Mutations are indicated as colored blocks, with whiteindicating no mutation, yellow indicating a mutant fraction of 0.1% to1%, orange indicate a mutant fraction of 1% to 10%, and red indicating amutant fraction of >10%.

FIG. 5 . Table 1. Epidemiology of Ovarian and Endometrial Tumors. Theestimated numbers of new cases and deaths in the U.S. from the majorsubtypes of ovarian and endometrial cancers are listed.

FIG. 6 . Table 2. Genetic Characteristics of Ovarian and EndometrialCancers. The frequencies of the commonly mutated genes in ovarian andendometrial cancers are listed.

FIG. 7 . Table S1. Endometrial Cancers (Endometrioid Subtype) Studied byWhole-exome Sequencing. The summary characteristics of the 22 cancersused for exome sequencing are listed.

FIG. 8 . Table S3. Mutations Assessed in Pap Smears. Clinicalcharacteristics of the 46 tumor samples are listed, along with themutation identified in each case and the fraction of mutant allelesidentified in the Pap smears.

FIG. 9 . Table S4. Primers Used to Assess Individual Mutations in PapSmears. The sequences of the forward and reverse primers used to testeach mutation via Safe-SeqS are listed in pairs (SEQ ID NO: 4-99,respectively).

FIG. 10 . Table S5. Primers Used to Simultaneously Assess 12 Genes inPap Smears. The sequences of the forward and reverse primers for eachtested region are listed in pairs (SEQ ID NO: 100-191, respectively).

FIG. 11 . Table S6. Mutations Identified in Pap Smears throughSimultaneous Assessment of 12 Genes. The fraction of mutant allelesidentified in the Pap smears using this approach is listed, along withthe precise mutations identified.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have developed a test for detecting different cancersusing samples that are already routinely collected for diagnosinguterine cancer and HPV (human papilloma virus) infection. Using a panelof genes, a high level of detection of both endometrial and ovariancancers was achieved.

Certain genes have been identified as mutated in a high proportion ofendometrial and ovarian cancers. These include CTNNB1, EGFR, PI3KCA,PTEN, TP53, BRAF, KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A,MLL2, RFF43, and FGFR2. The test can be performed on at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of these genes. Inaddition, other genes can be added or substituted into the panel toachieve a higher rate of detection.

Testing for a mutation may be done by analysis of nucleic acids, such asDNA or mRNA or cDNA. The nucleic acid analytes are isolated from cellsor cell fragments found in the liquid PAP smear sample. Suitable testsmay include any hybridization or sequencing based assay. Analysis mayalso be performed on protein encoded by the genes in the panel. Anysuitable test may be used including but not limited to massspectrometry. Other suitable assays may include immunological assays,such as, immunoblotting, immunocytochemistry, immunoprecipitation,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),including sandwich assays using monoclonal or polyclonal antibodies.

Genetic changes which can be detected are typically mutations such asdeletions, insertions, duplications, substitutions (missense or nonsensemutations), rearrangements, etc. Such mutations can be detected interalia by comparing to a wild type in another (non-tumor) tissue or fluidof an individual or by comparing to reference sequences, for example indatabases. Mutations that are found in all tissues of an individual aregermline mutations, whereas those that occur only in a single tissue aresomatic mutations. Epigenetic changes can also be detected. These may beloss or gain of methylation at specific locations in specific genes, aswell as histone modifications, including acetylation, ubiquitylation,phosphorylation and sumoylation.

Tests may be done in a multiplex format, in which a single reaction potis used to detect multiple analytes. Examples of such tests includeamplifications using multiple primer sets, amplifications usinguniversal primers, array based hybridization or amplification, emulsionbased amplification.

While probes and primers may be designed to interrogate particularmutations or particular portions of a gene, mRNA, or cDNA, these may notbe separate entities. For example, probes and primers may be linkedtogether to form a concatamer, or they may be linked to one or moresolid supports, such as a bead or an array.

Kits for use in the disclosed methods may include a carrier for thevarious components. The carrier can be a container or support, in theform of, e.g., bag, box, tube, rack, and is optionallycompartmentalized. The kit also includes various components useful indetecting mutations, using the above-discussed detection techniques. Forexample, the detection kit may include one or more oligonucleotidesuseful as primers for amplifying all or a portion of the target nucleicacids. The detection kit may also include one or more oligonucleotideprobes for hybridization to the target nucleic acids. Optionally theoligonucleotides are affixed to a solid support, e.g., incorporated in amicroarray included in the kit or supplied separately.

Solid supports may contain one single primer or probe or antibody fordetecting a single gene, protein, mRNA, or portion of a gene. A solidsupport may contain multiple primers, probes, or antibodies. They may beprovided as a group which will interrogate mutations at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the genes of thedesired panel. The panel may be selected from or comprise CTNNB1, EGFR,PI3KCA, PTEN, TP53, BRAF, KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A,CDKN2A, MLL2, RFF43, and FGFR2.

Primer pairs may be used to synthesize amplicons of various sizes.Amplicons may be for example from 50, 60, 75, 100, 125, 150, 200, 140,180 bp in length. Amplicons may run up to 200, 250, 300, 400, 500, 750,1000 bp in length, as examples. The size of the amplicon may be limitedby the size and/or quality of the template retrieved from the liquid PAPsmear. Probes and primers for use in the invention may contain awild-type sequence or may contain a sequence of a particular mutant.

In one embodiment, the test can be performed using samples that arecollected over time. The test results can be compared for quantitativeor qualitative changes. Such analysis can be used after a potentiallycurative therapy, such as surgery.

Georgios Papanicolaou published his seminal work, entitled “Diagnosis ofUterine Cancer by the Vaginal Smear,” in 1943 (31). At that time, hesuggested that endocervical sampling could, in theory, be used to detectnot only cervical cancers but also other cancers arising in the femalereproductive tract, including endometrial carcinomas. The researchreported here moves us much closer to that goal. In honor ofPapanicolaou's pioneering contribution to the field of early cancerdetection, we have named the approach described herein as the “PapGene”test.

One of the most important developments over the last several years isthe recognition that all human cancers are the result of mutations in alimited set of genes and an even more limited set of pathways throughwhich these genes act (32). The whole-exome sequencing data we present,combined with previous genome-wide studies, provide a striking exampleof the common genetic features of cancer (FIG. 6 , Table 2). Through theanalysis of particular regions of only 12 genes (FIG. 11 , table S5), wecould detect at least one driver mutation in the vast majority of ninedifferent gynecologic cancers (FIG. 5 , Table 1). Though several ofthese 12 genes were tumor suppressors, and therefore difficult totherapeutically target, knowledge of their mutational patterns providesunprecedented opportunities for cancer diagnostics.

The most important finding in this paper is that significant amounts ofcells or cell fragments from endometrial and ovarian cancers are presentin the cervix and can be detected through molecular genetic approaches.Detection of malignant cells from endometrial and ovarian carcinomas incervical cytology specimens is relatively uncommon. Microscopicexamination cannot always distinguish them from one another, fromcervical carcinomas, or from more benign conditions. Our study showedthat 100% of endometrial cancers (n=24), even those of low grade, and41% of ovarian cancers (n=22), shed cells into the cervix that could bedetected from specimens collected as part of routine Pap smears. Thisfinding, in conjunction with technical advances allowing the reliabledetection of mutations present in only a very small fraction of DNAtemplates, provided the foundation for the PapGene test.

This study demonstrates the value of sensitive endocervical DNA testingbut there are many issues that need to be addressed before optimalclinical use is achieved. The test, even in its current format, appearsto be promising for screening endometrial cancer, as the data in FIG. 3show that even the lowest stage endometrial cancers could be detectedthrough the analysis of DNA in Pap smear fluid through Safe-SeqS.However, only 41% of ovarian cancers could be detected in Pap smearseven when the mutations in their tumors were known. In eight of the ninePap smears from ovarian cancer patients that contained detectablemutations, the mutant allele fractions were >0.1% and therefore withinthe range currently detectable by PapGene testing (FIG. 9 , table S3).Further improvements in the technology could increase the technicalsensitivity of the PapGene test and allow it to detect more ovariancancers. Other strategies to increase this sensitivity involve physicalmaneuvers, such as massaging the adnexal region during the pelvicexamination or by performing the PapGene test at specified times duringthe menstrual cycle. Development of an improved method of collection mayalso be able to improve sensitivity. The current liquid specimen isdesigned for the detection of cervical cancer and as such utilizes abrush that collects cells from the ectocervix and only minimallypenetrates the endocervical canal. A small cannula that can beintroduced into the endometrial cavity similar to the pipelleendometrial biopsy instrument could theoretically obtain a more enrichedsample of cells coming from the endometrium, fallopian tube and ovary(33).

The high sensitivity and the quantitative nature of the PapGene testalso opens the possibility of utilizing it to monitor the response tohormonal agents (e.g., progestins) used to treat young women with lowrisk endometrial cancers. Some of these women choose to preservefertility, undergoing medical therapy rather than hysterectomy (34). Thedetection of pre-symptomatic ovarian cancers, even if advanced, couldalso be of benefit. Although not entirely analogous, it has beendemonstrated that one of the most important prognostic indicators forovarian cancer is the amount of residual disease after surgicaldebulking. Initially, debulking was considered optimal if the residualtumor was less than 2 cm. Subsequently, the threshold was reduced to 1cm and surgeons now attempt to remove any visible tumor. With eachimprovement in surgical debulking, survival has lengthened (35). A smallvolume of tumor is likely to be more sensitive to cytotoxic chemotherapythan the large, bulky disease typical of symptomatic high-grade serouscarcinoma.

An essential aspect of the screening approach described here is that itshould be relatively inexpensive and easily incorporated into the pelvicexamination. Evaluation of HPV DNA is already part of routine Pap smeartesting because HPV analysis increases the test's sensitivity (36, 37).The DNA purification component of the PapGene test is identical to thatused for HPV, so this component is clearly feasible. The preparation ofDNA, multiplex amplification, and the retail cost of the sequencingcomponent of the PapGene test can also be performed at a cost comparableto a routine HPV test in the U.S. today. Note that the increasedsensitivity provided by the Safe-SeqS component of the PapGene test (seeExample 6) can be implemented on any massively parallel sequencinginstrument, not just those manufactured by Illumina. With the reductionin the cost of massively parallel sequencing expected in the future,PapGene testing should become even less expensive.

There are millions of Pap smear tests performed annually in the U.S.Could PapGene testing be performed on such a large number of specimens?We believe so, because the entire DNA purification and amplificationprocess can be automated, just as it is for HPV testing. Though it maynow seem unrealistic to have millions of these sophisticatedsequence-based tests performed every year, it would undoubtedly haveseemed unrealistic to have widespread, conventional Pap smear testingperformed when Papanicolaou published his original paper (31). Eventoday, when many cervical cytology specimens are screened usingautomated technologies, a significant percentage require evaluation by askilled cyotpathologist. In contrast, the analysis of PapGene testing isdone completely in silico and the read-out of the test is objective andquantitative.

In sum, PapGene testing has the capacity to increase the utility ofconventional cytology screening through the unambiguous detection ofendometrial and ovarian carcinomas. In addition to the analysis of muchlarger numbers of patients with and without various types ofendometrial, ovarian, and fallopian tube cancers, the next step in thisline of research is to include genes altered in cervical cancer as wellas HPV amplicons in the multiplexed Safe-SeqS assay (FIG. 11 , tableS5). These additions will provide information that could be valuable forthe management of patients with the early stages of cervical neoplasia,as HPV positivity alone is not specific for the detection of cervicalcancer and its precursor lesions, particularly in young, sexually activewomen who frequently harbor HPV infections in the absence of neoplasia.

The above disclosure generally describes the present invention. Allreferences disclosed herein are expressly incorporated by reference. Amore complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only, and are not intended to limit the scope of theinvention.

EXAMPLE 1

We reasoned that more sophisticated molecular methods might be able todetect the presence of cancer cells in endocervical specimens at highersensitivities and specificities than possible with conventional methods.In particular, we hypothesized that somatic mutations characteristic ofendometrial and ovarian cancers would be found in the DNA purified fromroutine liquid-based Pap smears (henceforth denoted as “Pap smears”;FIG. 1 ). Unlike cytologically abnormal cells, such oncogenic DNAmutations are specific, clonal markers of neoplasia that should beabsent in non-neoplastic cells. However, we did not know if such DNAwould indeed be present in endocervical specimens, and we did not knowif they would be present in a sufficient amount to detect them. Theexperiments described here were carried out to test our hypothesis.

There were four components to this study: I. Determination of thesomatic mutations typically present in endometrial and ovarian cancers;II. Identification of at least one mutation in the tumors of 46 patientswith these cancers; III. Determination of whether the mutationsidentified in these tumors could also be detected in Pap smears from thesame patients; and IV. Development of a technology that could directlyassess cells from Pap smears for mutations commonly found in endometrialor ovarian cancers.

EXAMPLE 2 Prevalence of Somatically Mutated Genes in Endometrial andOvarian Cancers

There are five major histopathologic subtypes of ovarian cancers. Themost prevalent subtype is high grade serous (60% of total), followed byendometrioid (15%), clear cell (10%), and low-grade serous carcinoma(8%) (Table 1). Genome-wide studies have identified the most commonlymutated genes among the most prevalent ovarian cancer subtypes (Table 2)(23-25).

Such comprehensive studies have not yet been reported for theendometrioid and mucinous subtypes, collectively representing ˜20% ofovarian cancer cases (Table 1). However, commonly mutated genes in theendometrioid and mucinous subtypes have been reported (26). Inaggregate, the most commonly mutated gene in epithelial ovarian cancerswas TP53, which was mutated in 69% of these cancers (Table 2). Otherhighly mutated genes included ARID1A, BRAF, CTNNB1, KRAS, PIK3CA, andPPP 2R1A (Table 2).

Among endometrial cancers, the endometrioid subtype is by far the mostcommon, representing 85% of the total (Table 1). Because cancers of thissubtype are so frequent and have not been analyzed at a genome-widelevel, we evaluated them through whole-exome sequencing. The DNApurified from 22 sporadic endometrioid carcinomas, as well as frommatched non-neoplastic tissues, was used to generate 44 librariessuitable for massively parallel sequencing. The clinical aspects of thepatients and histopathologic features of the tumors are listed in table51. Though the examination of 22 cancers cannot provide a comprehensivegenome landscape of a tumor type, it is adequate for diagnosticpurposes—as these only require the identification of the most frequentlymutated genes.

Among the 44 libraries, the average coverage of each base in thetargeted region was 149.1 with 88.4% of targeted bases represented by atleast ten reads. Using stringent criteria for the identification ofsomatic mutations (as described in Materials and Methods), thesequencing data clearly demarcated the tumors into two groups: tencancers (termed the N Group, for non-highly mutated) harbored <100somatic mutations per tumor (median 32, range 7 to 50), while 12 cancers(termed the H Group, for highly mutated) harbored >100 somatic mutationsper tumor (median 674, range 164 to 4,629) (FIG. 7 , table S1).

The high number of mutations in the Group H tumors was consistent with adeficiency in DNA repair. Eight of the 12 Group H tumors hadmicrosatellite instability (MSI-H, table S1), supporting thisconjecture. Moreover, six of the Group H tumors contained somaticmutations in the mismatch repair genes MSH2 or MSH6, while none of theGroup N cancers contained mutations in mismatch repair genes. Mismatchrepair deficiency is known to be common among endometrial cancers andthese tumors occur in 19-71% of women with inherited mutations ofmismatch repair genes (i.e., patients with the Hereditary NonpolyposisColorectal Cancer) (27).

12,795 somatic mutations were identified in the 22 cancers. The mostcommonly mutated genes included the PIK3 pathway genes PTEN and PIK3CA(28), the APC pathway genes APC and CTNNB1, the fibroblast growth factorreceptor FGFR2, the adapter protein FBXW7, and the chromatin-modifyinggenes ARID1A and MLL2 (Table 2). Genes in these pathways were mutated inboth Group N and H tumors. Our results are consistent with prior studiesof endometrioid endometrial cancer that had evaluated small numbers ofgenes, though mutations in FBXW7, MLL2 and APC had not been appreciatedto occur as frequently as we found them. It was also interesting thatfew TP53 mutations (5%) were found in these endometrial cancers (Table2), a finding also consistent with prior studies.

Papillary serous carcinomas of the endometrium account for 10-15% ofendometrial cancers, and a recent genome-wide sequencing study of thistumor subtype has been published (29). The most common mutations in thissubtype are listed in Table 2. The least common subtype of endometrialcancers is clear cell carcinomas, which occur in <5%. Genes reported tobe mutated in these cancers were garnered from the literature (Table 2).

EXAMPLE 3 Identification of Mutations in Tumor Tissues

We acquired tumors from 46 cancer patients in whom Pap smears wereavailable. These included 24 patients with endometrial cancers and 22with ovarian cancers; clinical and histopathologic features are listedin table S3.

Somatic mutations in the 46 tumors were identified through whole-exomesequencing as described above or through targeted sequencing of genesfrequently mutated in the most common subtypes of ovarian or endometrialcancer (Table 2). Enrichment for these genes was achieved using a customsolid phase capture assay comprised of oligonucleotides (“captureprobes”) complementary to a panel of gene regions of interest. For theoncogenes, we only targeted their commonly mutated exons, whereas wetargeted the entire coding regions of the tumor suppressor genes.

Illumina DNA sequencing libraries were generated from tumors and theirmatched non-neoplastic tissues, then captured with the assay describedabove. Following amplification by PCR, four to eight captured DNAlibraries were sequenced per lane on an Illumina GA IIx instrument. Ineach of the 46 cases, we identified at least one somatic mutation (tableS3) that was confirmed by an independent assay, as described below.

EXAMPLE 4 Identification of Somatic Mutations in Pap Smears

In the liquid-based Pap smear technique in routine use today, theclinician inserts a small brush into the endocervical canal during apelvic exam and rotates the brush so that it dislodges and adheres toloosely attached cells or cell fragments. The brush is then placed in avial of fixative solution (e.g., ThinPrep). Some of the liquid from thevial is used to prepare a slide for cytological analysis or forpurification of HPV DNA. In our study, an aliquot of the DNA purifiedfrom the liquid was used to assess for the presence of DNA from thecancers of the 46 patients described above. Preliminary studies showedthat the fixed cells or cell fragments in the liquid, pelleted bycentrifugation at 1,000 g for five minutes, contained >95% of the totalDNA in the vial. We therefore purified DNA from the cell pellets whenthe amount of available liquid was greater than 3 mL (as occurs withsome liquid-based Pap smear kits) and, for convenience, purified DNAfrom both the liquid and cells when smaller amounts of liquid were inthe kit. In all cases, the purified DNA was of relatively high molecularweight (95%>5 kb). The average amount of DNA recovered from the 46 Papsmears was 49.3±74.4 ng/ml (table S3).

We anticipated that, if present at all, the amount of DNA derived fromneoplastic cells in the Pap smear fluid would be relatively smallcompared to the DNA derived from normal cells brushed from theendocervical canal. This necessitated the use of an analytic techniquethat could reliably identify a rare population of mutant alleles among agreat excess of wild-type alleles. A modification of one of theSafe-SeqS (Safe-Sequencing System) procedures described in (30) wasdesigned for this purpose (FIG. 2 ).

In brief, a limited number of PCR cycles was performed with a set ofgene-specific primers. One of the primers contained 14 degenerate Nbases (equal probability of being an A, C, G, or T) located 5′ to itsgene-specific sequence, and both primers contained sequences thatpermitted universal amplification in the next step. The 14 N's formedunique identifiers (UID) for each original template molecule. SubsequentPCR products generated with universal primers were purified andsequenced on an Illumina MiSeq instrument. If a mutation preexisted in atemplate molecule, that mutation should be present in every daughtermolecule containing that UID, and such mutations are called“supermutants” (30). Mutations not occurring in the original templates,such as those occurring during the amplification steps or through errorsin base calling, should not give rise to supermutants. The Safe-SeqSapproach used here is capable of detecting 1 mutant template among 5,000to 1,000,000 wild-type templates, depending on the amplicon and theposition within the amplicon that is queried (30).

We designed Safe-SeqS primers (table S4) to detect at least one mutationfrom each of the 46 patients described in table S3. In the 24 Pap smearsfrom patients with endometrial cancers, the mutation present in thetumor was identified in every case (100%). The median fraction of mutantalleles was 2.7%, and ranged from 0.01% to 78% (FIG. 3 and table S3).Amplifications of DNA from non-neoplastic tissues were used as negativecontrols in these experiments to define the detection limits of eachqueried mutation. In all cases, the fraction of mutant alleles wassignificantly different from the background mutation levels determinedfrom the negative controls (P<0.001, binomial test). There was noobvious correlation between the fraction of mutant alleles and thehistopathologic subtype or the stage of the cancer (FIG. 3 and tableS3).

In two endometrial cancer cases, two mutations found in the tumor DNAwere evaluated in the Pap smears (table S3). In both cases, themutations were identified in DNA from the Pap smear (table S3).Moreover, the ratios between the mutant allele fractions of the twomutations in the Pap smears were correlated with those of thecorresponding tumor samples. For example, in the Pap smear of case PAP083 the mutant allele fractions for the CTNNB1 and PIK3CA mutations were0.143% and 0.064%, respectively—a ratio of 2.2 (=0.14% to 0.064%). Inthe primary tumor from PAP 083, the corresponding ratio was 2.0 (79.5%to 39.5%).

Similar analysis of Pap smear DNA from ovarian cancer patients revealeddetectable mutations in nine of the 22 patients (41%). The fraction ofmutant alleles was smaller than in endometrial cancers (median of 0.49%,range 0.021% to 5.9%; see FIG. 3 and table S3). All but one of the caseswith detectable mutations were epithelial tumors; the exception was adysgerminoma, a malignant germ cell tumor of the ovary (table S3). Aswith endometrial cancers, there was no statistically significantcorrelation between the fraction of mutant alleles and histopathologiccriteria. However, most ovarian cancers are detected only at an advancedstage, and this was reflected in the patients available in our cohort.

EXAMPLE 5 A Genetic Test for Screening Purposes

The results described above document that mutant DNA molecules from mostendometrial cancers and some ovarian cancers can be found in routinelycollected Pap smears. However, in all 46 cases depicted in FIG. 3 , aspecific mutation was known to occur in the tumor, and an assay wassubsequently designed to determine whether that mutation was alsopresent in the corresponding Pap smears. In a screening setting, thereobviously would be no known tumor prior to the test. We thereforedesigned a prototype test based on Safe-SeqS that could be used in ascreening setting (FIG. 2 ).

This multiplexed approach included 50 primer pairs that amplifiedsegments of 241 to 296 bp containing frequently mutated regions of DNA.The regions to be amplified were chosen from the results described inSection I and included exons from APC, AKT1, BRAF, CTNNB1, EGFR, FBXW7,KRAS, PIK3CA, PPP2R1A, PTEN, and TP53. In control experiments, 46 of the50 amplicons were shown to provide information on a minimum of 2,500templates; the number of templates sequenced can be determined directlyfrom SafeSeqS-based sequencing (FIG. 2 ). Given the accuracy ofSafeSeqS, this number was adequate to comfortably detect mutationsexisting in >0.1% of template molecules (30). The regions covered bythese 46 amplicons (table S5), encompassing 10,257 bp, were predicted tobe able to detect at least one mutation in >90% of either endometrial orovarian cancers.

This test was applied to Pap smears of 14 cases—twelve endometrial andtwo ovarian—as well as 14 Pap smears collected from normal women. The 14cancer cases were arbitrarily chosen from those which had mutant allelefractions >0.1% (table S3) and therefore above the detection limit ofthe multiplexed assay. In all 14 Pap smears from women with cancer, themutation expected to be present (table S3) was identified (FIG. 4 andtable S6). The fraction of mutant alleles in the multiplexed test wassimilar to that observed in the original analysis of the same samplesusing only one Safe-SeqS primer pair per amplicon (table S3 and tableS6). Importantly, no mutations were detected in the 14 Pap smears fromwomen without cancer (FIG. 4 ; see Materials and Methods).

EXAMPLE 6 Materials and Methods Patient Samples

All samples for this study were obtained using protocols approved by theInstitutional Review Boards of The Johns Hopkins Medical Institutions(Baltimore, Md.), Memorial Sloan Kettering Cancer Center (New York,N.Y.), University of Sao Paulo (Sao Paulo, Brazil), and ILSbio, LLC(Chestertown, Md.). Demographic, clinical and pathologic staging datawas collected for each case. All histopathology was centrallyre-reviewed by board-certified pathologists. Staging was based on 2009FIGO criteria (38).

Fresh-frozen tissue specimens of surgically resected neoplasms of theovary and endometrium were analyzed by frozen section to assessneoplastic cellularity by a board-certified pathologist. Serial frozensections were used to guide the trimming of Optimal Cutting Temperature(OCT) compound embedded frozen tissue blocks to enrich the fraction ofneoplastic cells for DNA extraction.

Formalin-fixed paraffin embedded (FFPE) tissue samples were assessed bya board-certified pathologist (Propath LLC, Dallas, Tex.) for tumorcellularity and to demarcate area of high tumor cellularity. Tumortissue from serial 10 micron sections on slides from the original tumorblock were macrodissected with a razorblade to enrich the fraction ofneoplastic cells for DNA extraction.

The source of normal DNA was matched whole blood or non-neoplasticnormal adjacent tissue.

Liquid-based Pap smears were collected using cervical brushes andtransport medium from Digene HC2 DNA Collection Device (Qiagen) orThinPrep 2000 System (Hologic) and stored using the manufacturer'srecommendations.

Unless otherwise indicated, all patient-related values are reported asmean ±1 standard deviation.

DNA Extraction

DNA was purified from tumor and normal tissue as well as liquid-basedPap Smears using an AllPrep kit (Qiagen) according to the manufacturer'sinstructions. DNA was purified from tumor tissue by adding 3 mL RLTMbuffer (Qiagen) and then binding to an AllPrep DNA column (Qiagen)following the manufacturer's protocol. DNA was purified from Pap smearliquids by adding five volumes of RLTM buffer when the amount of liquidwas less than 3 mL. When the amount of liquid was >3 mL, the cells andcell fragments were pelleted at 1,000×g for five minutes and the pelletswere dissolved in 3 mL RLTM buffer. DNA was quantified in all cases withqPCR, employing the primers and conditions previously described (39).

Microsatellite Instability Testing

Microsatellite instability was detected using the MSI Analysis System(Promega), containing five mononucleotide repeats (BAT-25, BAT-26,NR-21, NR-24 and MONO-27) and two pentanucleotide repeat loci, per themanufacturer's instructions. Following amplification, the fluorescentPCR products were sized on an Applied Biosystems 3130 capillaryelectrophoresis instrument (Invitrogen). Tumor samples were designatedas follows: MSI-high if two or more mononucleotides varied in lengthcompared to the germline DNA; MSI-low if only one locus varied; andmicrosatellite stable (MSS) if there was no variation compared to thegermline. Pentanucleotide loci confirmed identity in all cases.

Preparation of Illumina DNA Libraries and Capture for Exomic Sequencing

Preparation of Illumina genomic DNA libraries for exomic and targetedDNA captures was performed according to the manufacturer'srecommendations. Briefly, 1-3 μg of genomic DNA was used for librarypreparation using the TruSeqDNA Sample Preparation Kit (Illumina). TheDNA was acoustically sheared (Covaris) to a target size of ˜200 bp. Thefragments were subsequently end-repaired to convert overhangs into bluntends. A single “A” nucleotide was then added to the 3′ ends of bluntfragments to prevent them from later self-ligation; a corresponding “T”on the 3′ end of adaptor molecules provided the complementary overhang.Following ligation to adaptors, the library was amplified with 8-14cycles of PCR to ensure yields of 0.5 and 4 μg for exomic and targetedgene captures, respectively.

Exomic capture was performed with the SureSelect Human Exome Kit V 4.0(Agilent) according to the manufacturer's protocol, with the addition ofTruSeq index-specific blocks in the hybridization mixture

(AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-XXXXX-ATCTCGTATGCCGTCTTCTGCTTGT (SEQID NO: 1), where the six base pair “XXXXXX” denotes one of 12sample-specific indexes).

Targeted Gene Enrichment

Targeted gene enrichment was performed by modifications of previouslydescribed methods (40, 41). In brief, targeted regions of selectedoncogenes and tumor suppressor genes were synthesized as oligonucleotideprobes by Agilent Technologies. Probes of 36 bases were designed tocapture both the plus and the minus strand of the DNA and had a 33-baseoverlap. The oligonucleotides were cleaved from the chip by incubatingwith 3 mL of 35% ammonium hydroxide at room temperate for five hours.The solution was transferred to two 2-ml tubes, dried under vacuum, andredissolved in 400 μL of ribonuclease (RNase)—and deoxyribonuclease(DNase)—free water. Five microliters of the solution was used for PCRamplification with primers complementary to the 12-base sequence commonto all probes: 5′-TGATCCCGCGACGA*C-3′ (SEQ ID NO: 2) and5′-GACCGCGACTCCAG*C-3′ (SEQ ID NO: 3), with * indicating aphosphorothioate bond. The PCR products were purified with a MinElutePurification Column (Qiagen), end-repaired with End-IT DNA End-RepairKit (Epicentre), and then purified with a MinElute Purification Column(Qiagen). The PCR products were ligated to form concatamers as described(40).

The major difference between the protocol described in (40, 41) and theone used in the present study involved the amplification of the ligatedPCR products and the solid phase capture method. The modifications wereas follows: 50 ng of ligated PCR product was amplified using the REPLI-gMidi Kit (Qiagen) with the addition of 2.5 nmol Biotin-dUTP (Roche) in a27.5 μL reaction. The reaction was incubated at 30° C. for 16 hours, thepolymerase was inactivated at 65° C. for 3 mins. The amplified probeswere purified with QiaQuick PCR Purification Columns (Qiagen). Forcapture, 4-5 μg of library DNA was incubated with 1 μg of the preparedprobes in a hybridization mixture as previously described (40). Thebiotinylated probes and captured library sequences were subsequentlypurified using 500 μg Dynabeads® MyOne Streptavidin (Invitrogen). Afterwashing as per the manufacturer's recommendations, the capturedsequences were eluted with 0.1 M NaOH and then neutralized with 1MTris-HCl (pH 7.5). Neutralized DNA was desalted and concentrated using aQIAquick MinElute Column (Qiagen) in 20 μL. The elute was amplified in a100 μL Phusion Hot Start II (Thermo Scientific) reaction containing1×Phusion HF buffer, 0.25 mM dNTPs, 0.5 μM each forward and reverseTruSeq primers, and 2 U polymerase with the following cyclingconditions: 98° C. for 30 s; 14 cycles of 98° C. for 10 s, 60° C. for 30s, 72° C. for 30 s; and 72° C. for 5 min. The amplified pool containingenriched target sequences was purified using an Agencourt AMPure XPsystem (Beckman) and quantified using a 2100 Bioanalyzer (Agilent).

Next-Generation Sequencing and Somatic Mutation Identification

After capture of targeted sequences, paired-end sequencing using anIllumina GA IIx Genome Analyzer provided 2×75 base reads from eachfragment. The sequence tags that passed filtering were aligned to thehuman genome reference sequence (hg18) and subsequent variant-callinganalysis was performed using the ELANDv2 algorithm in the CASAVA 1.6software (Illumina). Known polymorphisms recorded in dbSNP were removedfrom the analysis. Identification of high confidence mutations wasperformed as described previously (24).

Assessment of Low-Frequency Mutations

Primer Design. We attempted to design primer pairs to detect mutationsin the 46 cancers described in the text. Primers were designed asdescribed (30), using Primer3. (42) Sixty percent of the primersamplified the expected fragments; in the other 40%, a second or thirdset of primers had to be designed to reduce primer dimers ornon-specific amplification.

Sequencing Library Preparation. Templates were amplified as describedpreviously (30), with modifications that will be described in fullelsewhere. In brief, each strand of each template molecule was encodedwith a 14 base unique identifier (UID)—comprised of degenerate N bases(equal probability of being an A, C, G, or T)—using two to four cyclesof amplicon-specific PCR (UID assignment PCR cycles, see FIG. 2 ). Whileboth forward and reverse gene-specific primers contained universal tagsequences at their 5′ ends—providing the primer binding sites for thesecond-round amplification—only the forward primer contained the UID,positioned between the 5′ universal tag and the 3′ gene-specificsequences (four N's were included in the reverse primer to facilitatesequencing done on paired-end libraries) (table S4). The UID assignmentPCR cycles included Phusion Hot Start II (Thermo Scientific) in a 50 μLreaction containing 1×Phusion HF buffer, 0.25 mM dNTPs, 0.5 μM each offorward (containing 14 N's) and reverse primers, and 2 U of polymerase.Carryover of residual UID-containing primers to the second-roundamplification, which can complicate template quantification (30), wasminimized through exonuclease digestion at 370 C to degradeunincorporated primers and subsequent purification with AMPure XP beads(Beckman) and elution in 10 μL TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

The eluted templates were amplified in a second-round PCR using primerscontaining the grafting sequences necessary for hybridization to theIllumina GA IIx flow cell at their 5′ ends (FIG. 2 ) and two terminal 3′phosphorothioates to protect them from residual exonuclease activity(30). The reverse amplification primer additionally contained an indexsequence between the 5′ grafting and 3′ universal tag sequences toenable the PCR products from multiple individuals to be simultaneouslyanalyzed in the same flow cell compartment of the sequencer (30). Thesecond-round amplification reactions contained 1×Phusion HF buffer, 0.25mM dNTPs, 0.5 μM each of forward and reverse primers, and 2 U ofpolymerase in a total of 50 4. After an initial heat activation step at980 C for 2 minutes, twenty-three cycles of PCR were performed using thefollowing cycling conditions: 980 C for 10 s, 650 C for 15 s, and 720 Cfor 15 s. The multiplexed assay was performed in similar fashionutilizing six independent amplifications per sample with the primersdescribed in table S5. The PCR products were purified using AMPure XPbeads and used directly for sequencing on either the Illumina MiSeq orGA IIx instruments, with equivalent results.

Data Analysis. High quality sequence reads were analyzed as previouslydescribed. (30) Briefly, reads in which each of the 14 bases comprisingthe UID (representing one original template strand; see FIG. 2 ) had aquality score ≥15 were grouped by their UID. Only the UIDs supported bymore than one read were retained for further analysis. Thetemplate-specific portion of the reads that contained the sequence of anexpected amplification primer was matched to a reference sequence setusing a custom script (available from the authors upon request).Artifactual mutations—introduced during the sample preparation and/orsequencing steps—were eliminated by requiring that >50% of reads sharingthe same UID contained the identical mutation (a “supermutant;” see FIG.2 ). For the 46 assays querying a single amplicon, we required that thefraction of mutant alleles was significantly different from thebackground mutation levels determined from a negative control (P<0.001,binomial test). As mutations are not known a priori in a screeningenvironment, we used a more agnostic metric to detect mutations in themultiplexed assay. A threshold supermutant frequency was defined foreach sample as equaling the mean frequency of all supermutants plus sixstandard deviations of the mean. Only supermutants exceeding thisthreshold were designated as mutations and reported in FIG. 4 and tableS6.

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The disclosure of each reference cited is expressly incorporated herein.

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1-25. (canceled)
 26. A method comprising: a) testing a liquid Pap specimen obtained from a human subject for at least one of the following nucleic acid mutations: i) FBXW7 1435C>T, ii) PIK3CA 323G>A, iii) TP53 328C>T, iv) PIK3CA 263G>A, and v) CTNNB1 100G>A; and b) detecting the presence of at least one of the following: i) the T allele at position 1435 of FBXW7 gene; ii) the A allele at position 323 of PIK3CA gene; iii) the T allele at position 328 of TP53 gene; iv) the A allele at position 263 of PIK3CA gene; and v) the A allele at position 100 of CTNNB1 gene.
 27. The method of claim 26, wherein said human subject has an endometrial tumor.
 28. The method of claim 26, wherein said human subject has endometrial cancer.
 29. The method of claim 26, wherein the liquid Pap specimen is collected from the cervix.
 30. The method of claim 26, wherein the liquid Pap specimen comprises cells from the ectocervix.
 31. The method of claim 26, wherein the presence of the T allele at position 1435 of FBXW7 gene is detected.
 32. The method of claim 26, wherein the presence of the A allele at position 323 of PIK3CA gene is detected.
 33. The method of claim 26, wherein the presence of the T allele at position 328 of TP53 gene is detected.
 34. The method of claim 26, wherein the presence of the A allele at position 263 of PIK3CA gene is detected.
 35. The method of claim 26, wherein the presence of the A allele at position 100 of CTNNB1 gene is detected. 